Selective quantitative N-functionalization of unprotected α-amino acids using NHC-Ir(III) catalyst

Summary Unnatural amino acids are valuable building blocks with numerous applications. Here, we present a quantitative technique for accessing mono-N-functionalized amino acids directly from unprotected substrates using alcohols as alkylating agents and an NHC-Ir(III) catalyst. We detail specific steps for catalyst preparation and application, as well as for catalyst recycling. The protocol excludes a few amino acids (l-cysteine, l-lysine, and l-arginine) and secondary alcohols. For complete details on the use and execution of this protocol, please refer to Bermejo-López et al. (2022).1

Here, we present a quantitative technique for accessing mono-N-functionalized amino acids directly from unprotected substrates using alcohols as alkylating agents and an NHC-Ir(III) catalyst. We detail specific steps for catalyst preparation and application, as well as for catalyst recycling. The protocol excludes a few amino acids (L-cysteine, L-lysine, and L-arginine) and secondary alcohols. For complete details on the use and execution of this protocol, please refer to Bermejo-Ló pez et al. (2022). 1

BEFORE YOU BEGIN
Background N-Substituted amino acids are commonly used as chiral building blocks for the synthesis of pharmaceuticals, 2,3 biodegradable surfactants 4 and ligands for asymmetric catalysts, 5,6 among others. However, previous synthetic methodologies to obtain these compounds from unprotected amino acids present several limitations due to the nature of these subtrates. 7 Amino acids have limited solubility in non-polar organic solvents, and their zwitterionic nature makes them sensitive to pH changes and basic/acidic reagents. 8,9 N-Alkylation of unprotected amino acids is problematic since competing esterification of the carboxylic moiety may take place. Moreover, their high polarity complicates the purification/separation from the unreacted starting materials or by-products, which is normally tackled by protection/purification/deprotection steps. This increases significantly the number of synthetic steps to obtain the target molecule, requiring longer times and unnecessary use of resources.
To this date, there is only one methodology capable of N-alkylating unprotected amino acids with alcohols through hydrogen borrowing strategy in good yields and excellent retention of the optical purity. 10 However, double N-alkylations were obtained in most of the cases and further derivatization steps were needed to purify the products that failed to be formed in quantitative yields.
This protocol describes an iridium(III)-catalyzed selective mono-N-alkylation of unprotected L-phenylalanine with benzyl alcohol. The method is based in our recent work 1 where more than 100 chiral amino acids were N-functionalized with outstanding efficiency and with no need for further derivatization or purification steps (Scheme 1).
The preparation of the NHC-Ir(III) catalyst is described within this protocol, as well as the mono-Nalkylation of L-phenylalanine. The method can be expanded to a broad scope of amino acids described in Bermejo-Ló pez et al. 1

MATERIALS AND EQUIPMENT
Nuclear magnetic resonance (NMR) spectra were recorded at 400 or 500 MHz for 1 H NMR, and at 100 or 125 MHz for 13 C NMR, on a Bruker 400 and on a Bruker AV 500 spectrometer. 1 H NMR spectra were recorded using a relaxation delay T1 = 5 s (important integral regions of spectra with T1 > 5 s were equal to integral regions when T1 = 5 s). High-resolution mass spectra (HRMS) were obtained on a Bruker MicroTOF ESI-TOF spectrometer. Enantiomeric excess was determined using SFC-DIAD 250 mm Chiralcel or Chiralpak columns, CO 2 /CH 3 OH, using chiral stationary phases. Hydrogenation reaction performed in Parr Stainless Steel High Pressure Reactor Autoclave 1 L. The synthesis of NHC-Ir(III) pre-catalyst (Ir-1) is shown in Scheme 2. From imidazole (1), two consecutive alkylations, first with 2-bromoacetophenone (2) followed by n-butyl chloride, gave imidazolium salt 4. Oxime 5 is obtained in nearly quantitative yield in the following step, which is then reduced using H 2 in the presence of Pd/C. Imidazolium salt 6 is used in the next step without further purification. A silver carbene is formed upon reaction with Ag 2 O, which afterward is transmetalated with [Cp*IrCl 2 ] 2 to give the thermally and air stable pre-catalyst iridium complex (Ir-1). a. Weight out imidazole (1.36 g, 20 mmol) and 2-bromoacetophenone (4.77 g, 24 mmol, 1.2 equiv.) and add to a 100 mL round-bottom flask. b. Add 50 mL of acetone and Et 3 N (2.8 mL, 24 mmol, 1.0 equiv.) at room temperature with constant stirring (300-400 rpm; 20 C-23 C). c. Introduce the mixture in a pre-warmed oil bath at 50 C (under reflux) for 2 h.

STEP-BY-STEP METHOD DETAILS
Note: a white precipitate immediately forms. d. Cool down the mixture to room temperature (20 C-23 C) and filter-off the white precipitate; wash it with acetone (20 mL 3 3).
Note: the white solid can be analyzed by 1 H NMR (DMSO-d 6 ) observing a mixture of the triethylammonium salt and the corresponding salt derived from the alkylated product.
e. Dry the remaining solution containing the desired product 3 over MgSO 4 (2 g) and evaporate the solvent under reduced pressure in the rotavapor (40 C, 500 mbar). f. Purify by column chromatography using acetone as eluent. Imidazole 3 is obtained as a yellowish solid (1.61 g, 43% yield).
Note: analyze the crude by TLC with acetone as eluent. The product has an R f = 0.5.
i. Dry loading (dissolve compound 3 in the minimum amount of acetone (20-30 mL), add silica (0.5-1 g) to the mixture, remove the solvent under reduced pressure and add the silica containing compound 3 to the column) to a silica gel column (⌀ = 4 cm, height of silica added = 15 cm).
Note: silica is loaded and packed in the column using acetone as solvent. No airflow is used once the product is loaded in the column.
ii. Use TLC (acetone as eluent) with UV-light detection (254 nm) to analyze the fractions.
iii. Collect the fractions containing the product (R f = 0.5).
Note: discard the first dark yellow fraction. After this, the product comes out (be careful in the last tubes taken since imidazole 1 comes next). 2. Synthesis of imidazolium salt 4 ( Figure 2). a. In a 30 mL microwave tube, dissolve compound 3 (1.7 g, 9.13 mmol) in dry acetonitrile (7 mL). b. To the same vessel, add n-butylchloride (2.9 mL, 27.39 mmol, 3.0 equiv.). c. Close the vessel with the corresponding aluminum cap and heat it at 100 C for 24 h.
Note: oil bath already warmed at the corresponding temperature.
Note: analyze the crude by TLC with CH 2 Cl 2 /MeOH (5:1) as eluent. The product has an R f = 0.5.
i. Dry loading to a silica gel column (⌀ = 4 cm, height of silica added = 17 cm).
Note: silica is loaded and packed in the column using CH 2 Cl 2 /MeOH (20:1) as solvent. No airflow is used once the product is loaded in the column.
ii. Use TLC (CH 2 Cl 2 /MeOH 5:1 as eluent) with UV-light detection (254 nm) to analyze the fractions. iii. Collect the fractions containing the product (R f = 0.5). Note: start with CH 2 Cl 2 /MeOH 20:1 until the first dark brown fraction comes out, then change to CH 2 Cl 2 /MeOH 10:1 and continue until you do not see any impurity with high R f . Finally, use CH 2 Cl 2 /MeOH 5:1 to elute the product. f. Add H 2 O (3 mL) and a solution of NH 3 in H 2 O (25%; 1.62 mL, 1.2 equiv.) dropwise while stirring (10-15 min; 300-400 rpm). g. Remove the solvent at reduced pressure in a rotavapor. h. Dissolve the oily product 5 in the minimum amount of methanol (5-10 mL). i. Add CH 2 Cl 2 until the solution becomes cloudy (ca 7-10 mL). j. Store the mixture in the freezer (À18 C) for 15-30 min. k. Filter-off the solution (containing the desired product) to remove the unwanted white solid.
Wash the solid with CH 2 Cl 2 (15 mL 3 3).    (MeOD-d 4 ). Full conversion to imidazolium salt 6 is observed, which is used without further purification for the next step. CRITICAL: Suspending the Pd/C catalyst in the hydrogenation solution containing oxime 5 should be done under inert atmosphere. Further, Pd/C can spark spontaneously and may ignite on exposure to air after the hydrogenation reaction. Therefore, the reactor must be purged with inert atmosphere before is opened to air at the end of the reaction. The hydrogenation reaction must be performed inside a fume hood and the pressure of hydrogen released slowly.
Note: avoid exposure to light as much as possible in all the steps. The septum of the tube is removed to add Ag 2 O. The septum is put back and an additional purge with argon must be done. The septum is then exchanged fast by a crimp cap.  Note: after the reaction, exposure to light is no longer an issue.
i. Load the iridium crude mixture dissolved in the minimum amount of CH 2 Cl 2 (2-5 mL) with a pipette to a silica gel column (⌀ = 4 cm, height of silica added = 17 cm).
Note: silica is loaded and packed in the column using CH 2 Cl 2 /MeOH (20:1) as solvent. No airflow is used once the product is loaded in the column.
iii. Collect the fractions containing the product (R f = 0.2).
Note: start with CH 2 Cl 2 /MeOH 20:1 (300 mL, three times), then use CH 2 Cl 2 /MeOH 10:1 until the end. The two yellowish and orange first fractions of the column (Figure 6 -3 rd picture) should be discarded. Ir-1 comes out in the last yellow fraction of the column.
g. Remove the solvent of the combined fractions under reduced pressure in the rotavapor. h. Dissolve the residue with the minimum amount of CH 2 Cl 2 (5-10 mL) and add pentane until a yellow precipitate is formed. i. Remove the solvent by decantation and dry the solid Ir-1 under high vacuum. Ir-1 is obtained as a yellow solid (215 mg, 40% yield).
CRITICAL: AgNTf 2 salt is highly hygroscopic and sensitive to light, it must be stored in a desiccator and protected from light. Do not keep it in the fridge/freezer.
Note: The reaction does not require inert atmosphere.
c. Seal the microwave vial and stir the mixture at 90 C for 20 h.
Note: if after a few hours an off-white precipitate does not form, the activation of Ir-1 failed. Troubleshooting 3.

EXPECTED OUTCOMES
This protocol employs an efficient NHC-Ir(III) catalyst to achieve mono-N-alkylation of unprotected a-amino acids in a single step by using alcohols as alkylating agents. The applicability has been proven by synthesizing a large variety of N-alkylated amino acids with high retention of stereochemistry. 1 The targeted modified amino acids were obtained in quantitative yields after a simple filtration without the need of derivatization or further purification techniques.

LIMITATIONS
This protocol excludes amino acids containing a thiol group as cysteine (Cys), as well as basic amino acids such as lysine (Lys) and arginine (Arg). Another limitation of the methodology was encountered using secondary alcohols as alkylating agents.

Problem 1
The synthesis of the silver carbene 7 is a critical step in which the non-exposure to light and inert atmosphere are important (step 5).

Potential solution
To ensure formation of silver carbene 7, check crude by 1 H NMR (CDCl 3 ) before setting the next transmetalation step (Figure 10). Important to check the disappearance of the signal at 8.97 (s, 1H) from compound 6.

Problem 2
After collecting and evaporating the fractions corresponding to Ir-1 catalyst, the complex is dissolved with the minimum amount of CH 2 Cl 2 and precipitated with pentane. However sometimes after this, Ir-1 should be precipitated/purified again repeating this precipitation process with pentane (step 6).

Potential solution
Here you can see and compare the 1 H NMR (CDCl 3 ) spectra of the impure Ir-1 complex and the purified Ir-1 (Figure 11).

Problem 3
If the AgNTf 2 salt is not properly kept or manipulated or if the activation step fails, the N-alkylation does not work ( Figure 12) (step 8).

Potential solution
Keep AgNTf 2 in a desiccator and avoid exposure to light. Do not keep it in the fridge/freezer.

Potential solution
Product 10 solid was not washed efficiently. Further washing of 10 with diethyl ether is performed to ensure complete recovery of Ir-2.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Belé n Martín-Matute (belen.martin.matute@su.se).